To identify the signaling pathway that mediates the adrenergic stimulation of the expression of the gene for vascular endothelial growth factor (VEGF) during physiologically induced angiogenesis, we examined mouse brown adipocytes in primary culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced VEGF expression 3-fold, in a dose-and time-dependent manner (EC 50 Ϸ 90 nM). Also, the hypoxia-mimicking agent cobalt, as well as serum and phorbol ester, induced VEGF expression, but the effect of NE was additive to each of these factors, implying that a separate signaling mechanism for the NE-mediated induction was activated. The NE effect was abolished by propranolol and mimicked by isoprenaline or BRL-37344 and was thus mediated via ␤-adrenoreceptors. The NE-induced VEGF expression was fully cAMP mediated, an effect which was inhibited by H-89 and thus was dependent on protein kinase A activity. Involvement of other adrenergic signaling pathways (␣ 1 -adrenoreceptors, Ca 2؉ , protein kinase C, ␣ 2 -adrenoreceptors, and pertussis toxinsensitive G i -proteins) was excluded. The specific inhibitor of Src tyrosine kinases, PP2, markedly reduced the stimulation by NE, which demonstrates that a cAMP-dependent Src-mediated pathway is positively connected to VEGF expression. However, inhibition of Erk1/2 MAP kinases by PD98059 was without effect. NE did not prolong VEGF mRNA half-life and its effect was thus transcriptional, and was independent of protein synthesis. These results demonstrate that adrenergic stimulation, through ␤-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway that branches off from the NE-activated Src-Erk1/2 cascade to enhance transcription of the VEGF gene.

Male salmon exhibit alternative mating strategies, as both older anadromous adults and precocious juveniles (parr) participate in the spawning of a single female. This study tested the following hypotheses: 1) different intensities of sperm competition may reflect different sperm tail optima; 2) long spermatozoa are superior to short ones, with an associated cost on sperm longevity; and 3) a disfavored role in sperm competition selects for parr investing more in sperm quality. Comparisons included sperm morphological traits, whereas sperm quality was investigated by motility duration observations, measurement of the sperm adenylate system, and fertilization experiments. No evidence of different adaptive sperm dimensions between the male types was found. Positive association between spermatocrit and energy charge was, however, detected. Sperm length parameters correlated positively with ATP, energy charge, and fertilization success, whereas no evidence for an effect of sperm morphology on longevity was found. Male parr had greater spermatocrit than adults and fertilized equal proportions of eggs as adults despite a pronounced numerical subordinance in the fertilization experiments. It is concluded that a long sperm tail and midpiece may be selected to optimize energetic demands under conditions of increased sperm competition intensity.

)). Thus, the kinetics is such that respiration in the presence of physiological levels of NO is very sensitive to decreasing O2 tension.

To explain the distinctive pharmacological profiles observed for adrenergic stimulation of cell proliferation (beta1) and cell differentiation (beta3), the adrenergic control of cAMP accumulation was investigated during brown adipocyte development. In preadipocytes, norepinephrine (NE) increased cAMP levels but the beta3-agonists BRL-37344 and CGP-12177 did not; in contrast, when the cells had differentiated into mature brown adipocytes, a large cAMP response to the beta3-agonists had emerged and was now double that to NE (although the affinity of NE had increased 10-fold). Beta1-messenger RNA (mRNA) levels were high in both pre- and mature brown adipocytes; beta3-mRNA did not appear until maturation but then abruptly. Although beta1-receptors remained detectable by [3H]CGP-12177 binding in the mature brown adipocytes, the cAMP response to NE (based on propranolol inhibitory potency) switched from beta1 to beta3. Even the established beta1-agonist dobutamine acted through beta3-receptors in the mature brown adipocytes. The increases in cAMP levels could adequately explain the increased cell proliferation in NE-stimulated preadipocytes and the NE-induced UCP1 gene expression in mature brown adipocytes. The distinctive adrenergic profiles for stimulation of proliferation and of differentiation were thus not due to the existence of additional pathways but to a switch in the type of beta-receptor mediating the NE response, coordinated with an alteration in the nuclear response to increased cAMP levels. The study implies that full recruitment of brown adipose tissue cannot be induced by exclusive beta3-stimulation.

The mechanism behind the distinctive non-MichaelisMenten, bell-shaped kinetics of cAMP accumulation in brown adipocytes (which underlies the similar kinetics of UCP1 and ␤ 1 -adrenoreceptor gene expression) was investigated. A theoretical dual component analysis indicated that the observed dose-response curves could be constructed as the resultant of a stimulatory and an inhibitory component. Experimentally, inhibition of the ␣ 1 -component of the norepinephrine response revealed the underlying existence of a much larger stimulatory ␤ 3 -component which displayed monophasic MichaelisMenten kinetics. The inhibitory ␣ 1 -component (which was also monophasic but had a 2-fold higher EC 50 ) was mediated via an increase in [Ca 2؉ ] i ; the protein kinase C pathway was not involved. The [Ca 2؉ ] i increase which resulted in massive inhibition of cAMP accumulation was very low: <100 nM. The [Ca 2؉ ] i signal stimulated a calmodulin-controlled phosphodiesterase, possibly PDE-1. The acquirement of this specific interaction pattern between ␤-and ␣ 1 -adrenergic stimulation was thus part of the differentiation program of the brown adipocytes. It was concluded that an array of synergistic or inhibitory ␣ 1 /␤ interactions occur in the adrenergic regulation of this cell type which is unique in its dependence upon adrenergic stimulation for cellular proliferation, differentiation, and metabolic function.When stimulated with norepinephrine, brown adipocytes express the gene for the uncoupling protein-1 (UCP1) 1 (1-4). However, the kinetics of the induction of the expression of this gene do not adhere to simple, monophasic Michaelis-Menten kinetics. Rather, a distinctive bell-shaped response is observed, with norepinephrine concentrations of Ϸ0.1 M being optimal for induction of expression, and higher, as well as lower, concentrations being less efficient (3, 4).The observation of non-Michaelis-Menten kinetics such as these may easily be considered an experimental artifact and/or interpreted as an indication of "overstimulation" of the relevant receptor, leading to an acute "desensitization" of the response. Because of difficulties in determining kinetic parameters in non-Michaelis-Menten-responding systems, the downward part of such dose-response curves may often be ignored (or perhaps even omitted) in the presentation of data.However, in a theoretical analysis, Rovati and Nicosia (5) demonstrated that one conceivable possibility for generation of bell-shaped dose-response curves would be that these curves represent the resultant of interacting stimulatory and inhibitory components. They also implied that in such systems, large underlying stimulatory components, masked by the inhibitory component, could be revealed.Following this proposal, we have here attempted to resolve the bell-shaped dose-response curve for norepinephrine into dual components. As the kinetics of UCP1 gene expression have been demonstrated to mirror those of norepinephrineinduced cAMP accumulation in these cells (6), we have, de facto, examined whether spe...

To investigate a possible physiological desensitization process for beta 3-adrenergic responses, the effect of cold acclimation of hamsters on adrenergically stimulated oxygen consumption of isolated brown fat cells was investigated. Cells were prepared from control and from cold-acclimated hamsters. In agreement with earlier findings, cells isolated from cold-acclimated hamsters responded to norepinephrine addition with a decreased sensitivity (approximately 10 times higher 50% effective concentration) and a decreased maximal rate of oxygen consumption compared with cells from control hamsters. When cells were stimulated with the general beta-adrenergic agonist isoprenaline or with the beta 3-selective agonists BRL-37344 or CGP-12177, a similarly desensitized response was observed, demonstrating that it was indeed a beta 3-adrenergic response that was functionally desensitized. However, when the mitochondria within the cells were directly stimulated with exogenous free fatty acids (palmitate or octanoate), no difference between cells from control and cold-acclimated animals was seen, indicating that a mediatory step must be desensitized. When the cells were stimulated with forskolin (to activate adenylyl cyclase) or with 8-bromoadenosine 3',5'-cyclic monophosphate, the desensitized response was still observed. At post-adenosine 3',5'-cyclic monophosphate levels, a desensitization was not evident. Cyclic nucleotide phosphodiesterase activity was increased in cells from cold-acclimated animals. It is therefore suggested that this increased activity of phosphodiesterase could be (at least partly) responsible for the physiologically induced desensitized responses observed here.(ABSTRACT TRUNCATED AT 250 WORDS)

To explain the distinctive pharmacological profiles observed for adrenergic stimulation of cell proliferation (beta1) and cell differentiation (beta3), the adrenergic control of cAMP accumulation was investigated during brown adipocyte development. In preadipocytes, norepinephrine (NE) increased cAMP levels but the beta3-agonists BRL-37344 and CGP-12177 did not; in contrast, when the cells had differentiated into mature brown adipocytes, a large cAMP response to the beta3-agonists had emerged and was now double that to NE (although the affinity of NE had increased 10-fold). Beta1-messenger RNA (mRNA) levels were high in both pre- and mature brown adipocytes; beta3-mRNA did not appear until maturation but then abruptly. Although beta1-receptors remained detectable by [3H]CGP-12177 binding in the mature brown adipocytes, the cAMP response to NE (based on propranolol inhibitory potency) switched from beta1 to beta3. Even the established beta1-agonist dobutamine acted through beta3-receptors in the mature brown adipocytes. The increases in cAMP levels could adequately explain the increased cell proliferation in NE-stimulated preadipocytes and the NE-induced UCP1 gene expression in mature brown adipocytes. The distinctive adrenergic profiles for stimulation of proliferation and of differentiation were thus not due to the existence of additional pathways but to a switch in the type of beta-receptor mediating the NE response, coordinated with an alteration in the nuclear response to increased cAMP levels. The study implies that full recruitment of brown adipose tissue cannot be induced by exclusive beta3-stimulation.

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